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Regular Article |
a Department of Biology, University of Southern Colorado, Pueblo, Colorado 81001
b Department of Animal Health & Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53711
ABSTRACT
The goal of the present study was to investigate proteinase activity in uterine flushates collected during the zona loss time window (68–80 h post-egg activation) in both pregnant and pseudopregnant hamsters and in culture medium conditioned by hatching blastocysts. Several prominent enzyme activities appeared in all pregnant and pseudopregnant uterine flushates. However, only a 45, 43 x 10-3 Mr doublet coincided with the zona loss time window; these bands were absent outside of this time window and were not found in conditioned medium. In medium conditioned by hatching blastocysts, enzyme activity was represented by a 70, 65 x 10-3 Mr doublet identical to a doublet seen in all uterine flushates collected and in serum. There were 12 pregnant and 8 pseudopregnant uterine flushates that were capable of zona lytic activity in vitro (positive bioassays). Of these positive bioassays, five pregnant and four pseudopregnant uterine flushates exhibited the 45, 43 x 10-3 Mr doublet (correlative positive bioassays). These data suggest that there is an important uterine contribution to blastocyst escape from the zona pellucida, consisting of proteinases secreted during a finite time window prior to blastocyst attachment that are different from the proteinases responsible for the zona lytic activity in vitro.
conceptus, developmental biology
INTRODUCTION
The term hatching has been used in the literature to describe the process of zona loss. However, hatching is a misnomer because while most of the information currently available comes from in vitro studies, there are sufficient contradictory data from in vivo investigations to indicate that hatching is an artifact of embryo culture [1–5]. Under culture conditions, during the zona loss process, the blastocoele cavity expands with concomitant stretching and thinning of the zona pellucida, sometimes followed by collapse of the blastocysts with re-expansion. Eventually, there is focal rupture of the zona pellucida, and the embryo extrudes through this rupture, leaving the empty zona behind. These hatching events have been described in numerous species including the mouse [6, 7], pig [8], cow [9, 10], monkey [11], guinea pig [12–15], and human [16, 17]. In the hamster, however, in vitro zona loss occurs in a different way [1]. The blastocyst creates a focal area of lysis in the zona then emerges [18] through the hole as collapsed or expanded blastocysts. Perona and Wassarman [19] described a trypsin-like proteinase produced by the trophectoderm that assisted in the focal lysis or rupture of the zona pellucida in mouse blastocysts in vitro. They termed this enzyme "strypsin." Sawada et al. [20] verified these observations and, utilizing a battery of proteinase inhibitors, identified this in vitro hatching enzyme as a trypsin-like serine proteinase. Another suggestion is that plasmin generated by the action of embryonic u-PA (urokinase-type plasminogen activator) on uterine plasminogen chemically alters the zona pellucida to facilitate zona loss [21]. Due to a dearth of data on zona loss in vivo, the process of blastocyst hatching in vitro has been tacitly accepted as representing a natural event in rodents [22, 23]. Although these studies are informative, the mechanism of zona loss in vivo is still poorly understood, and the relative contributions of active zona loss (embryo dependent) vs. zona lysis (uterine dependent) remain unclear.
In order to shed light on the mechanism of zona loss in utero, the present study was developed to provide additional data on the potential uterine contribution of zona lysis. The objectives of the present study were the following: 1) to develop a bioassay to determine if a uterine flushate is capable of zona lytic activity in vitro, 2) to employ a sensitive proteinase detection assay (zymography) that could lead to the separation of proteinase activity in uterine flushings, 3) to utilize zymography to determine if the expression kinetics of proteinase activity over time corresponds to the kinetics of the zona loss time window described by Gonzales and Bavister [1], and 4) to compare the lytic activity responsible for hatching in vitro with the putative zona loss proteinase activity in vivo.
MATERIALS AND METHODS
Animals
Sexually mature golden hamsters (Mesocricetus auratus) were maintained on a 14L:10D schedule (lights-on at 0600 h central standard time and off at 2000 h). Naturally cycling females (non-gonadotropin-stimulated) used in this study were between 3 and 5 mo of age and weighed 110 to 130 g. All males used were proven breeders, and subsets of these males were vasectomized. After proof of sterility, the vasectomized males were used to induce pseudopregnancy in females [23–25]. Our animal colony undergoes review and approval following U.S. Department of Agriculture guidelines by an institutional review board four times per year.
Experimental Design
Uterine flushates were collected from both pregnant and pseudopregnant hamster populations over the preimplantation time interval, from 66 to 80 h post-egg activation (PEA) (2200 h on the third day of pregnancy to 2400 h on the fourth day of pregnancy) [1]. The uteri of n = 3 to 5 females per hour increment were flushed during this time interval from both populations (flushates were not pooled). With each of these flushates, bioassays and protein assays were run, and enzyme activities were evaluated by zymography (nonpregnant cycling females were used as controls for zymography, and the pregnant controls consisted of females outside of the zona escape time window). In addition, in each of these females, the number of eggs, embryos, and corpus lutea (CL) were counted, and changes in zona morphology of these eggs and embryos were noted, which are indicative of in utero zona lytic activity. The diameter of the zona pellucida before and after incubation in uterine flushate in the bioassay (only a change of 
5 µm in the zona diameter was considered significant) and of protein concentrations in uterine flushates were compared by ANOVA [26]. Changes in the diameter of zona following the bioassay and of uterine protein concentrations were plotted over time. Finally, the enzyme activity in hatching blastocysts-conditioned medium in vitro was compared on the same gels as uterine flushates.
Collection and Culture of Blastocysts for Conditioned Medium
Embryo cultures were conducted as described by Bavister et al. [27]. Briefly, 40 to 60 embryos were cultured in 150-µl drops of gas-equilibrated (10% CO2, 5% O2, and 85% N2 at 37°C in 100% humidity) HECM-4 overlaid with 10 ml washed silicone oil (Sigma Chemical Co., St. Louis, MO) [22, 25]. Pregnant females were killed on the third day of pregnancy at approximately 1500–1600 h for blastocyst collection. The blastocysts were cultured until 25% to 50% of the blastocysts had undergone or were in the process of zona loss in vitro (approximately 100–102 h PEA) [27–29].
Following blastocyst culture, the zona loss-conditioned medium was collected into a 1.5-ml microcentrifuge tube, centrifuged at 800 rpm for 4 min, and the supernate was collected. A 10% solution of SDS (Sigma) was added to the supernate to bring the concentration of SDS to 2.5% of the total volume. This conditioned medium was then equally divided between two 0.5-ml microcentrifuge tubes and stored at -70°C for subsequent zymography.
Blastocyst Collection for Bioassay
To determine if the uterine flushate of pregnant females could dissolve the zona pellucida of blastocysts, zona pellucida-intact embryos that experienced no apparent zona lytic activity were collected late on the third to early on the fourth day of pregnancy (between 2300 and 0300 h, when zona loss occurs in utero [1]). Embryos were flushed from uterine horns with 4°C flush solution consisting of 100 mM NaCl, 0.05% sodium azide (w/v; Sigma), 5 µg/ml gentamycin (Sigma), and 50 mM 3-[N-morpholino] propanesulfonic acid (MOPs; Sigma) at pH = 7.0 (pH 7-MOPS; adjusted by addition of 1.0 M HCl; 275 ± 5 mOsmol). Blastocysts with intact zona pellucidae were washed twice with pH 7-MOPs and used the day of the experiment.
Uterine Flushate Collection for Bioassay
The uterine flushates were obtained by flushing each uterine horn with 0.5 ml of pH 7-MOPs. In a previous experiment this pH was determined to be optimal for demonstrating enzyme activity by zymography (data not shown). Flushates were collected from pregnant and pseudopregnant females each hour from 66 to 80 h PEA (see Experimental Design). At each hour, both uterine horns of three to five pregnant and pseudopregnant females were flushed. Uterine flushates were also collected from 50 to 51 h PEA pregnant females (third day pregnant controls; n = 4), and uterine flushates were collected during 0600–0700 h from nonpregnant females at each of the four cycle days (cycle day nonpregnant controls; n = 4 each day). After the animals were killed at the designated time for collections, the full length of each uterine horn was excised and placed directly on ice. To minimize blood contamination during flushate collection, the uterine blood vessels were dissected from each uterine horn and the horns were washed by spraying with cold pH 7-MOPs from a 1-ml syringe through a 30-gauge flushing needle (Becton-Dickinson, Rutherford, NJ). The needle was then inserted in one end of the uterine horn, the horn was clamped around the needle with forceps, and 0.5 ml of flush solution was slowly injected. While the solution was being injected, the free end of the horn was placed into a 1.5-ml microcentrifuge tube on ice, and only the first three drops of solution were collected (approximately 80–110 µl per uterine horn). In all animals killed, the ovaries were excised, and the CL counted. To insure adequate pregnant-endocrine status, females with 
4 CL were rejected from the study.
Bioassay
One 15-µl drop of each of the uterine flushates described above (minus SDS), and one 15-µl drop of a negative control solution (flush solution that had not been passed through a uterine horn) were placed into 60- x 15-mm petri dishes (Becton Dickinson, Lincoln Park, NJ). The drops were overlaid with silicone oil, and, from the blastocyst collection described above, one embryo with an intact zona was added to each drop. Digitized images of each embryo prior to incubation were generated using a personal computer (100 Pentium Gateway 2000, N. Sioux City, SD) equipped with a PCVISIONplus frame grabber (Imaging Technology Inc., Woburn, MA), Optimas image-processing software (BioScan Optimas Corp., Edmonds, WA), and a digital camera (Sony, Lakewood, CO) attached to a teaching microscope with a 10x objective (American Optical, Buffalo, NY). Embryos were then incubated in 10% CO2 in air with saturated humidity at 37°C for 40 h. Following incubation, computer-digitized images were generated for comparison with the images made preincubation to determine if there was any change in the zona. The pre- and postincubation digital images were compared on a Vivitron 1572 video monitor (Gateway, Tokyo, Japan) using Optimas software. In this study, to avoid error, only a change in diameter 5 µm between the pre- and postincubation zonae was considered positive.
Uterine Flushate Collection for Zymography
The uterine flushate solutions for zymography were collected as described in the Uterine Flushate Collection for Bioassay section. In a preliminary experiment, to ascertain if the collection of the first three drops from each uterine horn was sufficient for the detection of proteinase activity by zymography, single drops were collected sequentially into a series of six microcentrifuge tubes from one horn of a pregnant female. This sequential collection was run on a zymogram to determine when, during the sequential collection, proteinase activity would no longer be detected in the flushate (data not shown).
Following collection, flushates were centrifuged at 800 rpm for 4 min to pellet debris and embryos and/or eggs. The volume of the supernate was determined and after removal of 15 µl of flushate for the bioassay, an appropriate quantity of a 10% solution of SDS was added to bring the concentration of SDS to an estimated 2.5% of the total volume. The supernate + SDS was gently vortexed. The supernate was then equally aliquotted into three sterilized 0.5-ml microcentrifuge tubes and stored at -70°C until use. The remaining pellet was resuspended with 30 µl flush solution, and the contents were examined microscopically. The number and zona morphology of the eggs and/or embryos collected in the pellet were recorded.
Zymography
Discontinuous dual slab polyacrylamide gels (Mini-Protean II Dual Slab Cell; BioRad, Hercules, CA), and buffers were prepared according to Laemmli [30]. The gels were 7 cm long by 8 cm wide by 1.5 mm with 10 wells. Resolving gels consisted of 0.1% gelatin (Sigma) and 10% acrylamide (BioRad). The stacking gel consisted of 4% acrylamide. The gels were assembled according to the protocol of the BioRad Mini-Protean II instruction manual.
Samples were prepared by the addition of 30 µl of uterine flushate (supernate + SDS) to 4 µl of sample buffer that consisted of 3.2 ml of MilliQ-water, 1.0 ml of 0.5 M Trizma base (pH 6.8), 1.6 ml glycerol (Sigma), 1.6 ml of 10% SDS, and 0.2% bromophenol blue (BioRad). To each well, 32–34 µl of this sample + sample buffer solution was added. Electrophoresis of the dual gels was run at 8 mA/gel at 4°C for the first hour and increased to 16 mA for approximately 4–6 h. After electrophoresis, the gels were rinsed twice and then soaked for 3 h at room temperature in wash-buffer consisting of 2.5% Triton X-100 (Sigma), and 0.05% sodium azide (w/v) in 50 mM Trizma base (pH 7.8) to remove the SDS and renature proteins. The gels were then rinsed twice and incubated in incubation buffer that consisted of 1.9 mM CaCl2, 100 mM NaCl, 0.05% sodium azide, and 0.1 mM MgCl2 in 50 mM Trizma base (pH 7.0–7.1) for 40 h at 37°C.
The gels were rinsed two times with distilled water and stained with 0.1% Coomassie blue (w/v; BioRad) in fixative consisting of 40% methanol and 10% acetic acid for 12 to 24 h. The gels were then rinsed twice with distilled water and destained for 1–3 h until the proteinase bands were cleared of stain. Digitized images of the gels were generated with a Kodak digital system (Kodak Digital Scientific, Rochester, NY).
The protein content for each uterine flushate was obtained using a Pierce BCA-200 protein microassay kit (Pierce, Rockford, IL). To estimate the molecular weights of the bands with proteinase activity, the log of the molecular weight standards (broad range; BioRad) was taken vs. electrophoretic migration to fit a linear regression model [26].
RESULTS
Bioassay
The bioassay was designed to determine if zona lytic activity in uterine flushates, collected from pregnant and pseudopregnant females, could be detected in vitro using the natural substrate, the zona pellucida of intact blastocysts. Figure 1 represents positive bioassays where the embryo in panel a was recovered at 67 h PEA, and the embryo in panel c was collected at 69 h PEA. These embryos were placed in a pregnant uterine flushate (Fig. 1, a and b) and in a pseudopregnant uterine flushate (Fig. 1, c and d) both collected at 75 h PEA and incubated at 37°C for 40 h. Figure 1, a and c, represent embryos prior to incubation and Figure 1, b and d, represent the same embryos following incubation. The changes in zona diameter were 22.4 ± 0.3 µm and 29.4 ± 0.4 µm for pregnant (Fig. 1, a and b) and pseudopregnant flushates (Fig. 1, c and d), respectively (Fig. 1 and Table 1). There was a total of 12 positive bioassays in pregnant (n = 55) and 8 in pseudopregnant flushates (n = 54) collected over the time interval 66–80 h PEA (Table 1). Additional to the 75-h positive pregnant uterine flushate bioassay demonstrated in Figure 1, a and b, there were positive bioassays that occurred in pregnant uterine flushates: one at 66 h, two at 67 h, one at 70 and 71 h, four at 73 h, one at 74 h, and an additional positive at 75 h PEA (Table 1). There was a total of eight positive bioassays that occurred in pseudopregnant uterine flushates: one each at 67 and 68 h, and two each at 73, 74, and 75 h PEA (Table 1). Of the positive bioassays demonstrated in Table 1, there was a subset of bioassays that exhibited conformational enzyme activity by zymography (see Zymography below). These positive bioassays with conformational enzyme activity occurred one each at 70, 71, and 73 h, and two at 75 h PEA in pregnant uterine flushates and two each at 74 and 75 h PEA in pseudopregnant uterine flushates (Table 1).
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The diameters of the zona pellucida pre- and postincubation in uterine flushates were measured using digital imaging software (Optimas). These diameters were plotted over time from 66 to 80-h PEA where there were three to five bioassays per time point for each hour interval (Fig. 2). Figure 2 compares preincubation diameters (crosshatched bars) with the same zonae postincubation (solid bars) at each time point over the zona loss time interval. The mean diameter of pre- and postincubation zonae incubated in pregnant uterine flushates was 116.66 ± 1.0 and 121.25 ± 1.0 µm, respectively (n = 55; P = 0.04). The mean diameter of pre- and postincubation zonae incubated in pseudopregnant uterine flushates was 114.46 ± 1.0 and 118.15 ± 1.0 µm, respectively (n = 54; P = 0.03). There was no significant difference (P > 0.10) between the preincubation zona diameters of the pregnant and pseudopregnant groups and no significant difference (P = 0.18) between the postincubation zona diameters between these two groups. When excluding all of the embryos in Table 1 from the data set, the remaining embryos represent negative bioassays. Comparisons of zona diameter of negative bioassays pre- and postincubation demonstrated no significant difference among the remaining 43 embryos incubated in pregnant uterine flushates (P = 0.15) and among the remaining 46 embryos incubated in the pseudopregnant uterine flushates (P = 0.12). Finally, there was no change between pre- and postincubation zona diameters of embryos incubated in flushate vehicle controls (pH 7-MOPS that had not passed through a uterine horn; P > 0.30).
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Changes in Zona Morphology of Eggs and Embryos In Utero
During the uterine flushate collection protocol, embryos and eggs from pregnant and pseudopregnant females, respectively, were examined to demonstrate when zona lytic activity occurred in vivo. In this study, eggs from pseudopregnant females underwent zona loss with similar kinetics to those described in the study conducted by Gonzales and Bavister [1] (data not shown). Figure 3 is a typical example of intact and zona-escaped unfertilized eggs flushed from a single pseudopregnant female when the cohort of eggs was in the process of zona loss. Note that the zona loss process is not simultaneous within a single pseudopregnant female (Fig. 3); within a single cohort there are zona-intact eggs with various zona diameters together with zona-escaped eggs.
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Protein Assay
The concentrations of total protein in uterine flushates in both pregnant and pseudopregnant females, collected at hourly increments, from 66 to 80 h PEA, were analyzed. The protein assay demonstrated no significant difference between the patterns of protein content over time in each of three replicates (P > 0.10), including preliminary studies (data not shown). The general trend in the overall protein concentration patterns increased at the beginning of the zona loss time window in both pregnant and pseudopregnant uterine flushates (from 66 to 76 h PEA; P 0.50) and decreased at 77 and 79 h PEA in pregnant and pseudopregnant, respectively (P > 0.562 between these time points). This decrease in total protein concentration occurred when zona loss in utero was 90% [1].
Zymography
In both pregnant and pseudopregnant hamsters the prominent enzyme activities were estimated to be a 132 x 10-3 Mr singlet, 102 x 10-3 Mr singlet, 83 x 10-3 Mr singlet, a 70, 65 x 10-3 Mr doublet, a 45, 43 x 10-3 Mr doublet, and a very light 20 x 10-3 Mr singlet (Fig. 4b). The appearance of a distinctive 45, 43 x 10-3 Mr doublet appeared only within the zona loss time window when approximately 40% of the embryos in utero are undergoing zona loss according to Gonzales and Bavister [1]. This distinctive doublet was absent outside of this time window (pregnant controls outside of the zona loss time window) and completely absent in nonpregnant controls (nonpregnant cycle day controls, see Materials and Methods). This doublet (45, 43 x 10-3 Mr) correlates precisely with the positive bioassays in uterine flushates collected at 70, 71, 73, and two at 75 h PEA from pregnant and two each at both 74 and 75 h PEA in pseudopregnant flushates (Table 1) and are represented in Figures 4 and 5.
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The general banding patterns as shown by zymography (Figs. 4a and 5a), at 67, 68, and 69 h PEA in pregnant flushates, are representative of enzyme activity seen when there are no correlative positive bioassays (n = 55) and no 45, 43 x 10-3 Mr doublet. Likewise, the two lanes at 73 and one at 74 h PEA (Fig. 4b) and the 68 PEA (Fig. 5b) are representative of enzyme activity seen when there are no positive correlative positive bioassays in pseudopregnant flushates (n = 54; these specific samples are not represented in Table 1). These enzyme-banding patterns (with no correlative positive bioassay) demonstrate identical enzyme activity with identical molecular weights. This enzyme activity banding pattern was also demonstrated in plasma (data not shown).
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The molecular weights with enzyme activity in hatching blastocyst-conditioned medium were in a 70, 65 x 10-3 Mr doublet (Fig. 5, a and b). These Mr x 10-3 banding patterns were present in all uterine flushate samples collected and were also observed in blood plasma.
DISCUSSION
Evidence for a uterine contribution to zona loss was provided by Mintz [31] in an experiment using BALB/c mice. Embryos with a homozygous genotype that causes arrest of embryos at the morula stage were compared with viable blastocysts by flushing at progressively later times. Zonae of both types of embryos were lysed in utero; however, under culture conditions, only the viable blastocysts underwent zona loss [31]. Because the zona lytic activity appeared in the uterus at about the time attachment was occurring, Mintz [31] concluded that the putative lysin might function as an implantation-initiating factor. In contrast, data obtained with hamster embryos and unfertilized eggs in pregnant and pseudopregnant animals, respectively [1, 23], demonstrated loss of the zona by gradual expansion and thinning, clearly implicating a uterine lytic activity in zona loss. In a unilateral oviduct ligation experiment conducted by Orsini and McLaren [2], zona loss in the control uterine horn proceeded normally, but loss of the zona from blastocysts trapped in the oviduct was delayed. Orsini also demonstrated that loss of the zona in the hamster is strongly progesterone dependent [3, 4]. Pinsker et al. [32] found that the activity of a mouse uterine protease detected by casein proteolysis increased more than 100-fold between Day 1 and Day 3 of pregnancy, with the peak occurring on Day 3. However, substantial activity (36% of the peak) was already present in uterine fluid on Day 2, which lessens the likelihood of this enzyme being solely responsible for sudden zona loss on Day 3 but does not exclude it. It was also considered that the uterine enzyme was involved in attachment and implantation rather than zona loss. Denker [33] showed that rabbit uterine flushings contain proteolytic activity, as did the conditioned medium of hatching blastocysts. Secretion of the uterine enzyme was progesterone-dependent.
In the present study, the bioassay demonstrated zona lytic activity upon the natural substrate (the zona pellucida of zona-intact blastocysts) in uterine flushates collected from pregnant and from pseudopregnant hamsters. Changes in zona diameter and thickness following incubation at 37°C in uterine flushates were indicative of a positive bioassay representing enzyme activity in uterine secretions. A positive bioassay in pregnant flushates (when blastocysts are present) does not prove that the enzyme activity is uterine mediated; however, a positive bioassay in pseudopregnant flushate (when only unfertilized eggs are present) indicates a uterine zona lytic activity. The bioassay and zymography results from this study support these assumptions. When bioassays were positive, there were unique bands at 45 and 43 x 10-3 Mr (a doublet) demonstrated by zymography in the same uterine flushates. These unique banding patterns were present when 40% of the embryos in utero are undergoing or have undergone zona loss during a time interval described by Gonzales and Bavister [1] (the zona escape time window) and did not appear outside of this time window or in nonpregnant controls. Pregnant controls prior to the zona loss time window and pregnant controls directly after the zona loss time window did not demonstrate this unique enzyme activity at 45 and 43 x 10-3 Mr. All uterine flush solutions with this unique lytic banding pattern seen on zymography demonstrated positive bioassays. However, not all of the uterine flush samples that demonstrated positive bioassays had the correlative 45, 43 x 10-3 Mr doublets (Table 1).
There may be several reasons for this incomplete correlation between the bioassays and the appearance of this unique banding pattern such as variation between animals including slightly different animal weights, different ages, different mass of the uterine horns, slightly different stages of embryo development, and potentially different endocrine signal intensity, and as a result, variation in pregnancy or pseudopregnancy timing. Other reasons may be related to the uterine flush protocol and the enzyme detection assay. For example, each animal had uterine horns with different mass, and the volume collected by flushing the uterine horn was variable (from 110 to 140 µl per animal; data not shown), so the uterine contents were diluted to different degrees. Our enzyme detection assay may not be sensitive enough to demonstrate enzyme activity in the most dilute samples. The enzyme detection assay employed required SDS, and therefore, renaturation of the proteins was necessary. This could create circumstances where the results could provide some false negatives and no false positives, which is what our zymography data show. Despite these shortfalls, the unique lytic activity demonstrated in both pregnant and pseudopregnant flushates (the 45, 43 x 10-3 Mr doublets) is the only candidate detected that may be responsible for zona loss in the intact and pregnant animal. The appearance of the 45, 43 x 10-3 Mr doublet in pseudopregnant hamsters conclusively demonstrates that it is uterine mediated and not trophectoderm mediated, as would be the case if blastocysts were present or if these bands were present in cultures from hatching blastocysts.
It would be expected that the lytic activity responsible for zona escape in utero is tightly regulated. As a result, we expect that within individual animals the zona lytic activity would occur during a finite time interval during preimplantation development [1], perhaps 2–4 h or less. Tight regulation may be responsible for the elusive behavior of the enzyme activity demonstrated in this study (the 45, 43 x 10-3 Mr doublet). This regulation may also be responsible for not detecting the doublet on zymography in all of our positive bioassays.
The molecular weight of the in vitro lytic activity demonstrated in this study (the 70, 65 x 10-3 Mr doublet) is comparable to the molecular weight of strypsin reported by Perona and Wassarman [19]. Perona and Wassarman reported a trypsin-like proteinase (estimated to be 74 x 10-3 Mr) that was associated with hatching mouse blastocysts in vitro. However, these authors [19] did not report a doublet. Despite this, the doublet demonstrated in vitro, in this study, and strypsin could be similar or the same as they were both demonstrated in hatching-blastocysts conditioned medium although in different animal models. This banding pattern (the 70, 65 x 10-3 Mr doublet) was also found in blood. Therefore, we suggest that this enzyme activity, when demonstrated in uterine flushates, is a contamination of the uterine flush protocol. The presence of this banding pattern in hatching-blastocyst conditioned medium suggests that this activity may be responsible for implantation [1] or be a fail-safe mechanism for zona escape, or both.
The phenomenon of zona loss, by unfertilized eggs in pseudopregnant females, was similar to the zona loss process exhibited by blastocysts in pregnant females both in this study and as described by other authors [1, 5]. The zona loss process was not simultaneous within a single cohort; the zona loss mechanism was by global lysis of the zona rather than by focal lysis, and zona loss occurred during a finite period during preimplantation development, prior to the time when blastocysts begin to attach to uterine epithelia. Because zona loss occurs by the same mechanism in pseudopregnant as in pregnant hamsters, then the zona loss of eggs in pseudopregnant females may be essentially an in vivo natural bioassay for the enzyme activity responsible for zona loss in the intact and pregnant animals.
All of the other bands with enzyme activity, the 132 x 10-3 Mr singlet, 102 x 10-3 Mr singlet, 83 x 10-3 Mr singlet, as well as the 70, 65 x 10-3 Mr doublet, and the 20 x 10-3 Mr singlet were consistently found in all of the uterine flushates collected in this study. Despite the presence of these enzyme activities, there were 43 and 46 negative bioassays in pregnant and pseudopregnant uterine flushates, respectively. The status of embryos and eggs in flushates from these animals was examined, and zona loss was similar to the zona loss as described by Gonzales and Bavister [1] and Montag et al. [5], in both pregnant and pseudopregnant animals, strongly suggesting that these enzyme activities play no direct role in zona lysis at their concentration in utero. There was variability in the intensity of these lytic activities over the zona escape time window in pregnant, pseudopregnant, and nonpregnant controls. However, this variability of intensity did not have a consistent pattern. These banding patterns, with lytic activity, were also found in hamster plasma (data not shown). Because the appearance of these bands is demonstrated consistently within every uterine flushate and in plasma, we conclude that these enzyme banding patterns and their variable intensities are a result of the uterine flush collection protocol and could be plasma contributions to uterine fluid. An interesting observation was that an identical banding pattern with enzyme activity at 70 and 65 x 10-3 Mr also appeared in hatching-blastocyst-conditioned medium. Based upon the embryo culture protocol in chemically defined medium, the only conclusion that can be made regarding this enzyme activity (the 70, 65 x 10-3 Mr doublet) is the same conclusion that Perona and Wassarman [19] and Sawada et al. [20] made: this enzyme activity demonstrated in hatching-blastocyst-conditioned medium is embryo mediated; however, we disagree with their hypothesis that this is the zona escape or hatching enzyme. We suspect a different function for the enzyme activity seen in hatching-blastocyst-conditioned medium. This enzyme activity may be part of the plasminogen-plasmin system as described by Strickland et al. [34] and Sappino et al. [35]. Also, it has been demonstrated that the trophectoderm produces a u-PA [34, 35] and the u-PA receptor [36]. Together u-PA and the u-PA receptor may be responsible for tissue remodeling and/or the directed invasive behavior of trophectoderm [37]. In addition, because embryo development and specifically loss from the zona pellucida is delayed in vitro [1, 28], it would be reasonable to speculate that the zona loss process in vitro is actually an implantation event as suggested by Gonzales and Bavister [1]. If this is true, then the 70, 65 x 10-3 Mr doublet demonstrated in this study and the 74 x 10-3 Mr activity described by Perona and Wassarman [19] and Sawada et al. [20] are part of the implantation mechanism. Another suggestion is that plasmin generated by the action of embryonic u-PA on uterine plasminogen chemically alters the zona pellucida to facilitate zona loss [21] or is a backup mechanism for zona loss in utero. Because data on zona loss in vivo are scarce and/or have been ignored, the process of blastocyst hatching in vitro has been erroneously accepted in rodents as representing a natural event [1, 22, 23]. We hypothesize that the in vitro lytic activity is the means for the invasive behavior of trophectoderm, i.e., implantation, while the unique uterine-mediated 45, 43 x 10-3 Mr doublet described here is the primary mechanism for zona loss in utero.
ACKNOWLEDGMENTS
For editorial assistance during the final stages of this manuscript the authors are grateful to Barbara Jean Gonzales, Margaret Senator, and Dr. Sandra Bonetti. We also thank Damain Thrasher, Rana Gonzales, and Felicia Rodriguez for their technical assistance.
FOOTNOTES
First decision: 28 March 2000.
1 This study was funded by the National Institutes of Health Biomedical Research Support Grant Program S06 GM08197-17. Preliminary data from this study were presented at the 32nd annual meeting of the Society for the Study of Reproduction (Biol Reprod 1999; 60[suppl 1]:505). 
2 Correspondence: David Gonzales, Department of Biology, University of Southern Colorado, 2200 Bonforte Blvd., Pueblo, CO 81001. FAX: 719 549 2732; dgonz{at}uscolo.edu
Accepted: August 22, 2000.
Received: February 9, 2000.
REFERENCES
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 V. Quesada, L. M. Sanchez, J. Alvarez, and C. Lopez-Otin Identification and Characterization of Human and Mouse Ovastacin: A NOVEL METALLOPROTEINASE SIMILAR TO HATCHING ENZYMES FROM ARTHROPODS, BIRDS, AMPHIBIANS, AND FISH J. Biol. Chem., June 18, 2004; 279(25): 26627 - 26634. [Abstract] [Full Text] [PDF]
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 A. A. Goyeneche, V. Calvo, G. Gibori, and C. M. Telleria Androstenedione Interferes in Luteal Regression by Inhibiting Apoptosis and Stimulating Progesterone Production Biol Reprod, May 1, 2002; 66(5): 1540 - 1547. [Abstract] [Full Text]
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